Presentation Information
[P04-461]A Morphology Engineering Strategy to Improve the Manufacturability of Filamentous Fungal Cell Factories
○Hui Ting Chu1,2 (1. National University of Singapore (Singapore), 2. Singapore Institute of Food and Biotechnology Innovation (Singapore))
Keywords:
Fungal biotechnology,Fungal morphology engineering,Chemical genetics,Genetic engineering,CRISPR/Cas9
Filamentous fungi are widely used in industrial biotechnology due to their high secretory capacity and ability to produce diverse enzymes and biomolecules for the pharmaceuticals, energy, and food sectors. However, their broader industrial adoption is constrained by unfavorable rheological behaviour during submerged cultivation. Entanglement of elongated fungal hyphae leads to high culture viscosity, inefficient mixing, and poor oxygen and nutrient transfer, resulting in suboptimal fermentation yield and high processing cost.
In this study, we applied an integrated genetic engineering and chemical genetics strategy to engineer fungal morphology and improve fermentation scalability. A lysozyme-producing Aspergillus oryzae strain was subjected to CRISPR/Cas9-mediated deletion of morphology-related genes and to targeted chemical perturbation to systematically evaluate relationships among morphology, rheology, and protein titre.
Genetic screening of morphology-related genes uncovered ΔnsdC and Δfmg31 hyper-branching mutants. The ΔnsdC mutant exhibited dispersed mycelial clumps under submerged conditions [2], while Δfmg31 mutant (unpublished) retained compact pellet morphology comparable to the wild type. Chemical perturbation of the fmg31-associated pathway using drug A reproducibly induced freely dispersed mycelia growth, resulting in substantial culture viscosity reduction and improved biomass accumulation.
While lysozyme titres were not significantly altered in any of the mutants and chemically perturbed strains, the reduction in viscosity in drug A-treated strain represents a key advantage for industrial fermentation, since it directly improves mass transfer, reduces power input requirements, and enhances robustness at scale.
Solid-state NMR-based cell wall analysis combined with transcriptomic profiling highlighted structural and regulatory components responsible for morphology modulation, uncovering new gene targets for strain and process optimization.
This work demonstrates that morphology engineering through the application of drug A is effective in controlling bioprocess parameters, including culture viscosity and biomass output. By understanding the cellular processes affected by drug A from cell wall analysis and transcriptomics profiling, new gene targets are identified for rational morphology engineering.
Collectively, this work demonstrates that morphology engineering can be decoupled from productivity to specifically improve process performance. By enabling controllable viscosity reduction without compromising protein production, this approach offers a scalable and transferable strategy to enhance the manufacturability of filamentous fungal cell factories in industrial bioprocesses.
In this study, we applied an integrated genetic engineering and chemical genetics strategy to engineer fungal morphology and improve fermentation scalability. A lysozyme-producing Aspergillus oryzae strain was subjected to CRISPR/Cas9-mediated deletion of morphology-related genes and to targeted chemical perturbation to systematically evaluate relationships among morphology, rheology, and protein titre.
Genetic screening of morphology-related genes uncovered ΔnsdC and Δfmg31 hyper-branching mutants. The ΔnsdC mutant exhibited dispersed mycelial clumps under submerged conditions [2], while Δfmg31 mutant (unpublished) retained compact pellet morphology comparable to the wild type. Chemical perturbation of the fmg31-associated pathway using drug A reproducibly induced freely dispersed mycelia growth, resulting in substantial culture viscosity reduction and improved biomass accumulation.
While lysozyme titres were not significantly altered in any of the mutants and chemically perturbed strains, the reduction in viscosity in drug A-treated strain represents a key advantage for industrial fermentation, since it directly improves mass transfer, reduces power input requirements, and enhances robustness at scale.
Solid-state NMR-based cell wall analysis combined with transcriptomic profiling highlighted structural and regulatory components responsible for morphology modulation, uncovering new gene targets for strain and process optimization.
This work demonstrates that morphology engineering through the application of drug A is effective in controlling bioprocess parameters, including culture viscosity and biomass output. By understanding the cellular processes affected by drug A from cell wall analysis and transcriptomics profiling, new gene targets are identified for rational morphology engineering.
Collectively, this work demonstrates that morphology engineering can be decoupled from productivity to specifically improve process performance. By enabling controllable viscosity reduction without compromising protein production, this approach offers a scalable and transferable strategy to enhance the manufacturability of filamentous fungal cell factories in industrial bioprocesses.
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